Synthesis of Alkynyl Amino Acids and Peptides and Their Palladium-Catalyzed Coupling to Ferrocene

A method for attaching organometallics to the C-terminus of amino acids via a Pd-catalyzed two-step procedure is presented. Boc-protected enantiomerically pure amino acids 1 (a Phe, b Leu, c Met, d Ser) are reacted with 1,1-diethylpropargylamine to yield alkynyl amino acids 2. After reaction with (p-iodoanilido)ferrocene carboxylic acid 3 in the presence of 5 mol % PdCl2(PPh3)2/CuI ferrocene amino acids 4 are obtained in ca. 80% yield. The reaction does not require anhydrous conditions and tolerates functional groups such as amides, alcohols (Ser, 4d) or thioethers (Met, 4c). A complete characterization by multinuclear NMR (including 15N) is carried out. Cyclic voltammetry shows a reversible wave at about +190 mV (vs Fc/Fc+) independent of the nature of the attached amino acid. In the solid state, 2a forms a left-handed helix along the crystallographic c axis which is stabilized by hydrogen bonds as revealed by a single-crystal X-ray structure determination. A comparison of IR data in solution and the solid state suggests that hydrogen bonding is also important for the solid-state structures of ferrocene amino acids 4 but does not play a role in solution. The use of this methodology for peptide chemistry is demonstrated by labeling the dipeptide Boc-Met-Phe-OH at the C-terminus and the tripeptide Boc-Phe-Glu-Leu-OMe with ferrocene. The alkyne anchoring group in the tripeptide is introduced at the Cγ(Glu) atom at an early stage of the peptide synthesis and is not affected by subsequent deprotection and coupling reactions

Labeling of Peptides with Halocarbonyltungsten Complexes Containing Functional η²-Alkynyl Ligands

Two different seven-coordinate iodocarbonyltungsten complexes incorporating tris(pyrazolyl)borate ligands and η2-coordinated functionalized alkynes have been synthesized and characterized. [Tp*WI(CO)(η2-HCC(CH2)2CO2H)] (1), which has been characterized in the solid state by X-ray diffraction, was readily coupled to the hexa- and pentapeptides pseudo-neurotensin(8-13) and enkephalin in solid phase peptide synthesis (SPPS) to provide the N-terminal-labeled bioconjugates [Tp*WI(CO)(η2-HCC(CH2)2CO-NH-Lys-Lys-Pro-Tyr-Ile-Leu-OH)] (3) and [Tp*WI(CO)(η2-HCC(CH2)2CO-NH-Tyr-Gly-Gly-Phe-Leu-OH)] (4), respectively. [Tp*WI(CO)(η2-Fmoc-Pgl-OH)] (2, Pgl = propargylglycine) was utilized to synthesize the side-chain-labeled enkephalin derivative H2N-Tyr-Gly-[Tp*W(I)(CO)(η2-Pgl)]-Phe-Leu-OH (5). All new bioconjugates were comprehensively charaterized by HPLC, mass spectrometry, and multinuclear 1D and 2D spectroscopy. Their characteristic metal carbonyl IR band just over 1900 cm−1 and good stability against air and water make these Tp*W alkyne complexes valuable labels for biomolecules

<i>C</i>‑Terminal Acetylene Derivatized Peptides <i>via</i> Silyl-Based Alkyne Immobilization

A new Silyl-based Alkyne Modifying (SAM)-linker for the synthesis of <i>C</i>-terminal acetylene-derivatized peptides is reported. The broad scope of this SAM2-linker is illustrated by manual synthesis of peptides that are side-chain protected, fully deprotected, and disulfide-bridged. Synthesis of a 14-meric (KLAKLAK)₂ derivative by microwave-assisted automated SPPS and a one-pot cleavage click procedure yielding protected 1,2,3-triazole peptide conjugates are also described

The Mo(η-allyl)(CO)₂ Moiety as a Robust Marker Group in Bioorganometallic Chemistry. Unusual Crystal Structure of the Phenylalanine Derivative Mo(C₅H₄-CO-Phe-OMe)(η-allyl)(CO)₂

The MoCp(η-C3H5)(CO)2 (Cp = η-cyclopentadienyl) moiety is introduced as a new labeling group in bioorganometallic chemistry. The acid Mo(C5H4-CO2H)(η-C3H5)(CO)2 (2) was obtained from the reaction of MoCp(η-C3H5)(CO)2 (1) with BuLi and solid CO2 followed by aqueous workup. Coupling of 2 to amino acids with various complexity and C-terminal functionality by standard peptide chemistry methods yielded the amino acid derivatives Mo(C5H4-CO-AA-R)(η-C3H5)(CO)2, 3 (3a, AA = Phe, R = OCH3; 3b, AA = Leu, R = NH2; 3c, AA = Gly, R = OCH3). In addition, the dipeptide derivative Mo(C5H4-CO-Leu-Phe-OCH3)(η-C3H5)(CO)2 (4) was synthesized by reacting 2 with H-Leu-Phe-OCH3. All new compounds are characterized by elemental analysis, IR, MS, and NMR spectroscopy. X-ray analysis on 3a shows the unit cell to contain two independent molecules, A and B, which differ mainly by the orientation of the allyl and carbonyl groups with respect to the amino acid substituent on the Cp ring. Furthermore, an allyl-endo conformation for both A and B is observed. This is the first example of such a conformation in the crystal structure of a MoCp(C3H5)(CO)2 derivative. In solution, both the exo and endo isomer are present, as concluded from 1H NMR spectroscopy approximately in a 4:1 ratio. The activation barriers of interconversion were determined to be 62.7 ± 0.5 kJ mol-1 (3a) and 60.5 ± 0.5 kJ mol-1 (3c)

The Mo(η-allyl)(CO)₂ Moiety as a Robust Marker Group in Bioorganometallic Chemistry. Unusual Crystal Structure of the Phenylalanine Derivative Mo(C₅H₄-CO-Phe-OMe)(η-allyl)(CO)₂

The MoCp(η-C3H5)(CO)2 (Cp = η-cyclopentadienyl) moiety is introduced as a new labeling group in bioorganometallic chemistry. The acid Mo(C5H4-CO2H)(η-C3H5)(CO)2 (2) was obtained from the reaction of MoCp(η-C3H5)(CO)2 (1) with BuLi and solid CO2 followed by aqueous workup. Coupling of 2 to amino acids with various complexity and C-terminal functionality by standard peptide chemistry methods yielded the amino acid derivatives Mo(C5H4-CO-AA-R)(η-C3H5)(CO)2, 3 (3a, AA = Phe, R = OCH3; 3b, AA = Leu, R = NH2; 3c, AA = Gly, R = OCH3). In addition, the dipeptide derivative Mo(C5H4-CO-Leu-Phe-OCH3)(η-C3H5)(CO)2 (4) was synthesized by reacting 2 with H-Leu-Phe-OCH3. All new compounds are characterized by elemental analysis, IR, MS, and NMR spectroscopy. X-ray analysis on 3a shows the unit cell to contain two independent molecules, A and B, which differ mainly by the orientation of the allyl and carbonyl groups with respect to the amino acid substituent on the Cp ring. Furthermore, an allyl-endo conformation for both A and B is observed. This is the first example of such a conformation in the crystal structure of a MoCp(C3H5)(CO)2 derivative. In solution, both the exo and endo isomer are present, as concluded from 1H NMR spectroscopy approximately in a 4:1 ratio. The activation barriers of interconversion were determined to be 62.7 ± 0.5 kJ mol-1 (3a) and 60.5 ± 0.5 kJ mol-1 (3c)

Chiral Ferrocene Amines Derived from Amino Acids and Peptides: Synthesis, Solution and X-ray Crystal Structures and Electrochemical Investigations

For the recognition of all but the simplest naturally occurring molecules, electrochemical sensors based on ferrocene will certainly require chiral centers. To advance the necessary chemistry, this work describes the synthesis and properties of ferrocene derivatives of enantiomerically pure amino acids, peptides, and other chiral amines. Ferrocene aldehyde is condensed with amino acid esters to yield the corresponding Schiff bases 2, which are reduced by NaBH4 in methanol to the ferrocene methyl amino acids 3. An X-ray single-crystal analysis was carried out on the phenylalanine derivative 3a (monoclinic space group P21, a = 10.301(1) Å, b = 9.647(1) Å, c = 18.479(2) Å, β = 102.98(2)°, Z = 4). Further peptide chemistry at the C terminus proceeds smoothly as demonstrated by the synthesis of the ferrocene labeled dipeptide Fc-CH2-Phe-Gly-OCH3 5 (Fc = ferrocenyl ((η-C5H4)Fe(η-C5H5))). We also report the synthesis of the C,N-bis-ferrocene labeled tripeptide Phe-Ala-Leu and its electrochemical characterization. Starting from the enantiomerically pure ferrocene derivative 9, which was synthesized from ferrocene aldehyde and l-1-amino-ethylbenzene, two diastereomers 10 were obtained by peptide coupling with N-Boc protected d- and l-alanine. There was, however, only very little diastereomeric induction if 0.5 equiv of a racemic mixture of alanine were used. This suggests that amino acid activation rather than coupling is the rate-determining step. A combination of NOESY (nuclear Overhauser effect spectroscopy) spectra and molecular modeling furnished detailed insights into the solution structures of 3, 9, and 10 and was used to rationalize their different reactivity

Ligand Modifications on a Cp(quinolate)Ru Catalyst to Improve Its Stability in a Bio-orthogonal Deprotection Reaction

The deprotection or activation of substances in biological systems is of particular interest as this method can be used to activate prodrugs in a site- and time-specific manner, thus minimizing possible side effects. Investigations of the literature-known Ru catalyst [RuCp(QL)(η3-allyl)PF6] (with Cp = η-cyclopentadienide, QL= 5-(methoxycarboyl)-8-quinolinolate, 5c) revealed stability issues of the dissolved catalyst in air. We surmised that a more stable catalyst would perform better under biologically relevant conditions and that classical modifications in the ligand set would affect such improved properties. In this work, a systematic study is reported to modify the Cp ligand by using Cp* (Cp* = η-pentamethyl-cyclopentadienide), trimethylsilyl Cp, or t-butyl Cp instead and on the allyl ligand by introducing a methyl group at the middle carbon of 5c. Periodical 1H NMR measurements in DMSO-d6 were performed to monitor the stability of the complexes for longer periods in air, and the catalytic activity of the synthesized compounds was investigated by the deprotection of an alloxycarbonyl (alloc)-protected fluorescent coumarin dye, as monitored by an increase in fluorescence intensity. Modification of the allyl ligand had no effect on the stability, but modification of the Cp ligand was shown to affect the stability of the dissolved complex and, in the case of Cp*, significantly prolong it. As expected, the more stable catalysts are catalytically active for a longer period, but as the reaction rate is not as fast, slightly lower or similar overall yields as compared to the original complex were achieved. Preliminary MTT testing of the obtained complexes revealed IC50 values in the low micromolar range

“Four-Potential” Ferrocene Labeling of PNA Oligomers via Click Chemistry

The scope of the Cu(I)-catalyzed [2 + 3] azide/alkyne cycloaddition (CuAAC, click chemistry) as a key reaction for the conjugation of ferrocene derivatives to N-terminal functionalized PNA oligomers is explored herein (PNA: peptide nucleic acid). The facile solid-phase synthesis of N-terminal azide or alkyne-functionalized PNA oligomer precursors and their cycloaddition with azidoferrocene, ethynylferrocene, and N-(3-ethylpent-1-yn-3-yl)ferrocene-carboxamide (DEPA−ferrocene) on the solid phase are presented. While the click reaction with azidomethylferrocene worked equally well, the ferrocenylmethyl group is lost from the conjugate upon acid cleavage. However, the desired product was obtained via a post-SPPS conversion of the alkyne−PNA oligomer with azidomethylferrocene in solution. The synthesis of all ferrocene−PNA conjugates (trimer t3-PNA, 3, 4, 5, 6; 12mer PNA, 10 - t c t a c a a g a c t c, 11 - t c t a c c g t a c t c) succeeded with excellent yields and purities, as determined by mass spectrometry and HPLC. Electrochemical studies of the trimer Fc−PNA conjugates 3, 4, 5, and 6 with four different ferrocene moieties revealed quasi-reversible redox processes of the ferrocenyl redox couple Fc0/+ and electrochemical half-wave potentials in a range of E1/2 = −20 mV to +270 mV vs FcH0/+ (Fc: ferrocenyl, C10H9Fe). The observed potential differences ΔE1/2min are always greater than 60 mV for any given pair of Fc−PNA conjugates, thus allowing a reliable differentiation with sensitive electrochemical methods like e.g. square wave voltammetry (SWV). This is the electrochemical equivalent of “four-color” detection and is hence denoted “four-potential” labeling. Preparation and electrochemical investigation of the set of four structurally different and electrochemically distinguishable ferrocenyl groups conjugated to PNA oligomers, as exemplified by the conjugates 3, 4, 5, and 6, demonstrates the scope of the azide/alkyne cycloaddition for the labeling of PNA with electrochemically active ferrocenyl groups. Furthermore, it provides a PNA-based system for the electrochemical detection of single-nucleotide polymorphism (SNP) in DNA/RNA

Efficient Reagent-Saving Method for the N‑Terminal Labeling of Bioactive Peptides with Organometallic Carboxylic Acids by Solid-Phase Synthesis

Labeling of biomolecules with organometallic moieties holds great promise as a tool for chemical biology and for the investigation of biochemical signaling pathways. Herein, we report a robust and reproducible synthetic strategy for the synthesis of ruthenocenecarboxylic acid, giving the acid in 53% overall yield. This organometallic label was conjugated via solid-phase peptide synthesis in near-quantitative yield to a number of different biologically active peptides, using only 1 equiv of the acid and coupling reagents, thereby avoiding wasting the precious organometallic acid. This optimized method of stoichiometric N-terminal acylation was then also successfully applied to conjugating ferrocenecarboxylic acid and a novel organometallic Re^I(CO)₃ complex, showing the generality of the synthetic procedure